SPECIFIC AIMS The organ of Corti consists of four rows of sensory hair cells along the length of the cochlear duct. On the apical surface of each hair cell, F-actin-filled stereociliary bundles form a [unreadable]V[unreadable]-shaped structure. Invariably, the vertices of the stereociliary [unreadable]V[unreadable]s all point to the periphery of the cochlea (Fig. 1). The uniform orientation of stereocilia manifests a polarity that is parallel to the plane of the epithelium and known as planar cell polarity (PCP) (Fig. 1)1-3. Defects in the patterning of hair cells and stereocilia are responsible for many forms of deafness4,5. The long term goal of our research is to uncover mechanisms underlying the morphogenesis of the organ of Corti for a better understanding of the molecular basis of deafness and for new treatment of deafness. In addition, cellular polarity is a fundamental biological issue key to the development and function of multicellular organisms6. The organ of Corti represents the most distinctive form of PCP in vertebrates and promises a unique opportunity to reveal underlying mechanisms for cellular polarization and morphogenesis1. In the first funding period (August, 2002-present) of this research, we showed that the primordial organ of Corti undergoes cellular intercalations7,8 characteristic of convergent extension (CE)9, for cochlear extension and precise patterning of 4 rows of hair cells. We demonstrated that a conserved genetic pathway, the PCP pathway, regulates both cochlear CE and uniform orientation of stereocilia7,10,11. This proposal seeks to build on these results and further explore the underlying mechanism for PCP signaling. In particular, the kinocilium, a microtubule-based primary cilium that projects from the centrosome-derived basal body of the cell, is positioned at the vertex of the [unreadable]V[unreadable]-shaped stereocilia5 and appears to lead the polarity of stereocilia during development7,12. We will test the hypothesis that ciliary genes are involved in PCP signaling in regulating the polarity of stereocilia and CE of the cochlea. The following specific aims are to be achieved. Specific Aim 1 To test whether a ciliary gene Polaris is involved in PCP-regulated processes in the cochlea The ciliary gene Polaris13-15 encodes a component of the intraflagellar transport (IFT) proteins that are required for ciliogenesis16-18. We will analyze the role of Polaris as the first step toward the understanding of ciliary genes in PCP signaling. We will determine the localization of Polaris in the cochlea (Exp. 1.1); conditionally inactivate (CKO) Polaris in the ear19-21 (Exp. 1.2); and analyze morphologic defects (Exp. 1.3) and potential CE defects (Exp. 1.4) in Polaris CKO mice. To date, we have detected characteristic PCP defects in Polaris mutants. In addition, circularly shaped stereociliary bundles were also observed in Polaris mutants. Specific Aim 2 To determine genetic and physical interactions between Polaris and known PCP regulators Our preliminary data implicated Polaris in PCP regulation. We will further test whether Polaris is indeed involved in PCP regulation by detecting genetic interactions between Polaris and known PCP genes (Exp. 2.1). In addition, the hallmark of PCP signaling is polarized subcellular localization of a set of PCP proteins along the polarization axis 1-3,22. We will test PCP protein localization in Polaris CKO mice (Exp. 2.2) and Polaris localization in PCP mutants (Exp. 2.3) to determine the genetic epistasis22. We will test whether Polaris interacts directly with PCP proteins by co-immunoprecipitation (coIP) (Exp. 2.4) and co-localization (Exp. 2.5). Specific Aim 3 To dissect the molecular role for ciliary genes in PCP establishment and maintenance CKO of Polaris after establishment of PCP will allow us to test for the first time whether PCP signaling is required for the maintenance of PCP (Exp. 3.1). In addition, Polaris is also localized to ciliary basal bodies besides kinocilia axoneme, and we found a previously unrecognized polarization process for basal bodies during PCP. Since the basal body (centrosome) is the organization center for polarizing cytoskeletal microtubules 16-18, it is likely the determinant for the intrinsic polarity of hair cells. We will assess the role of Polaris in the polarization of basal bodies and the role of basal bodies in stereocilia polarity (Exp. 3.2), and examine cilia-independent role of Polaris by CKO of ciliary genes post regression of kinocilia (Exp. 3.3). Critical experiments will be repeated for another ciliary mutant, Kif3a CKO (Exp. 3.4). Specific Aim 4 To further explore a direct link between ciliary genes and PCP signaling We identified TBC1d19 (MGI:1914499) in a 2-hybrid screen with the C-terminal domain of a known PCP protein, Ltap/Vangl2. TBC1d19 has not been studies in any organism, but its highly conserved Drosophila homolog was identified in a screen for ciliary genes23. The identification of TBC1d19 through its interaction with a PCP protein provides an opportunity to reveal a direct link between ciliary genes and PCP signaling. We will confirm the expression of TBC1d19 in the cochlea during PCP (Exp. 4.1) and its interaction with Ltap/Vangl2 (Exp. 4.2), and examine its subcellular localization (Exp. 4.3) and its role in ciliogenesis (Exp. 4.4). Specific Aim 5 To assess a role for TBC1d19 in PCP regulation Functional roles of TBC1d19 in PCP signaling will be explored first by examination of CE during zebrafish gastrulation and neurulation (Exp. 5.1), and PCP in lateral line hair cells (Exp. 5.2) and inner ear hair cells (Exp. 5.3) in zebrafish treated with TBC1d19 morpholinos. In addition, a CKO allele of TBC1d19 will be generated in mice and functional analyses of TBC1d19 in the mouse inner ear will be carried out (Exp. 5.4).